Eukaryotic cells have multiple mechanisms to regulate mRNA quality. This regulation is generally altered in aged cells and is dysfunctional in several age-related diseases. In order to fully understand how mRNA regulation contributes to aging, a thorough understanding of the mechanisms of mRNA regulation is required. A new mRNA surveillance checkpoint has recently been identified in yeast that requires the SUMO protease Ulp1. SUMO (small ubiquitin related modifier) is a highly conserved small protein that is conjugated to many proteins throughout the cell. Modification by SUMO ("sumoylation") has a variety of effects that can be reversed by SUMO removal ("desumoylation") by the ULP SUMO proteases, including Ulpl The newly identified mRNA checkpoint prevents intron-containing pre-mRNAs from exiting the nucleus, where they can be translated into nonsense protein fragments that are toxic. The involvement of Ulp1 in this checkpoint suggests that desumoylation is a key process regulating this pathway. The broad objective of this project is to gain a molecular understanding of the mechanisms by which sumoylation and desumoylation regulate mRNA quality control. The findings from this study will identify new regulatory factors whose dysregulation likely contributes to aging. Using the budding yeast Saccharomyces cerevisiae as a model organism, two specific aims will be addressed. Aim 1 will characterize the roles of sumoylation and desumoylation in the pre-mRNA retention checkpoint by analyzing mutants defective for SUMO conjugation and removal to determine their requirement for preventing pre-mRNA export. In addition, double mutant analysis will be employed to establish a genetic scheme for pre-mRNA surveillance that positions the SUMO machinery with respect to other pathway components. The goal of Aim 2 is to identify sumoylated substrates involved in pre-mRNA surveillance and determine their regulatory significance in this pathway. Candidate substrate proteins will be screened for SUMO modification and tested to determine whether fusing SUMO to the protein can cause defective pre-mRNA retention. In addition, novel proteins involved in this pathway will be identified through a genetic screen for high-copy suppressors of a synthetic growth defect between an ulpl and an mRNA surveillance mutant. [unreadable] [unreadable] Relevance: Altered regulation of mRNA in the cell plays a role in aging and many age-related diseases, including Alzheimer's disease and the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS). The goal of this project is to understand how a newly identified nuclear envelope-associated mechanism regulates mRNA. The results will thus provide new information regarding molecular events that likely contribute to aging and disease. [unreadable] [unreadable] [unreadable]